Skin equivalents have many uses not only as a replacement for human or animal skin for skin grafting, but also as test skin for determining the effects of pharmaceutical substances and cosmetics on skin.
A major difficulty in pharmacological, chemical and cosmetic testing is the difficulties in determining the efficacy and safety of the products on skin. One advantage of the skin equivalents of the invention is their use as an indicator of the effects produced by such substances through in vitro testing on test skin.
Also, skin grafting of denuded areas, of granulating wounds and of burns, still present major healing problems despite advances in grafting techniques. Split thickness autografts and epidermal autografts (cultured autogenic keratinocytes) have been used with variable success. However, both forms of treatment have many disadvantages. For example, split-thickness autografts are generally unavailable in large body surface area (BSA) burns, cause further injury to the patient, are of limited use in the treatment of patients with Dystrophic Epidermolysis bullosa (DEB), show limited tissue expansion, require repeated surgical operations and protracted hospitalization and give rise to undesirable cosmetic results. Epidermal autografts require time to be produced, have a low success ("take") rate of between 30-50%, often form spontaneous blisters, are fragile and difficult to handle, exhibit contraction to 60-70% of their original size, are vulnerable during approximately the first 15 days after grafting and are of virtually no use in the treatment of deep burns where both the dermis and epidermis have been destroyed.
An alternative form of treatment is epidermal allografts (cultured allogenic keratinocytes). American researchers have treated patients with second degree burns by grafting epidermal allografts onto wounds with some success. The benefits of such an allograft include: a ready supply of such grafts can be maintained so that the patients might be covered in a single procedure with a material which allows permanent healing to occur, it eliminates autografting which increases the area of wounds and leaves painful infection-prone donor sites, burn wounds covered with cultured allografts heal as quickly as burn wounds that have been covered with autografts, and enables the treatment of patients with DEB.
However, epidermal allografts still experience many of the limitations of epidermal autografts.
Full thickness skin injuries from burns destroy both the epidermis and dermis, and treatment With cultured skin needs to replace both of these components.
Hansborough, J. F and Boyce, S. T (JAMA 1989, 2125-2130) reported the application of auto epidermal cells onto a dermal equivalent which is then grafted onto a wound. The main disadvantage of this method lies in the preparation of the dermal equivalent.
Furthermore, this method involved the use of chondroitin-6-sulfate (GAG) which has weak bonding to the collagen at neutral pH, and thus may be released into the wound environment causing unforeseen long term effects on human subjects. GAG has been reported to increase scar formation in wounds which is something to be avoided in grafts. Another effect of GAG containing collagen sponges consisting in reduction of collagen blood clotting capacity can be considered rather unfavourable for application in bleeding wounds. Fibrin clot contributes to an adhesion of the graft to the wound.
Also, in this method, the collagen sponge is stabilized by being crosslinked with with 0.25% glutaraldehyde (GTA). Such crosslinked collagen is resorbed at a slower rate and is resistant to bacterial or fungal infection. At the same time the ingrowth of cells, infiltrating the GTA crosslinked collagen matrix-is less. Collagen crosslinked with GTA may retain this agent as a high molecular weight polymer which is continuously hydrolyzed and monomeric GTA is released and detactable for up to 6 weeks. The cytotoxic effect of GTA on fibroblasts in tissue culture suggests that it is not an ideal crosslinking agent for a dermal equivalent which is infiltrated by hosts cells and in which the bovine collagen matrix is rapidly degraded thus releasing GTA into body fluids.
Recently, living skin equivalent grafts comprising a dermal layer of rat fibroblast cells cast in soluble collagen and a epidermal layer of cultured rat keratinocytes were successfully grafted as allografts onto Sprague Dawley rats by Bell et.al. (Journal Investigative Dermatology,1983; 81:2s-10s). Histological examination of the graft revealed that the epidermal layer had fully differentiated to form desmosomes, tonofilaments, keratohyalin and a basement lamella. However, attempts to reproduce the living skin equivalent using human fibroblasts and keratinocytes met only with limited success. The keratinocytes failed to fully differentiate to form a basement lamella and the dermo-epidermal function was a straight line.
Problems of epidermalizing the surface of the above dermal equivalent resulted in Bell modifying his method by using skin biopsies as a source of keratinocytes, as described in the International PCT application, published as WO 86/02273. The problems with this approach are that the skin substitute thus produced is not uniform over its entire surface, since the biopsy, including the dermal portion, remains inserted in the dermis substitute. Also, the surface-area of the skin substitute obtained is limited by the number of biopsies that may be taken from a single person. The biopsy taking involve a medical procedure with potential problem of infection and scar formation. The punch biopsies of skin equivalent as a means of expanding the production of additional skin equivalent is a time consuming process. As a result, such a skin equivalent has not been used clinically. In 1988, a paper in the journal "Burns" by Colounb et al reported a 100% failure rate using Bell's method.
Bernard et al, the inventors of Australian Patent Application AU-A-13743/88 try to avoid the above problems by utilizing the culturing capacity of keratinocytes contained in the sheath of a hair follicle. The skin biopsy is replaced by a hair follicle enclosed in its sheath, which is implanted in a perpendicular position in the free surface of dermis substitute being formed. The main criticism of the above invention is the nature of the dermis and the fact that the period of epidermal growth may take several months.
Thus, there is a need for the development of living skin equivalent grafts comprising both the epidermal and dermal layers that can be easily prepared and maintained in sufficient quantities to enable a single treatment of skin wounds.
In developing a living skin equivalent it is desirable that it comprise at least some or all of the following features: it should enable rapid and sustained adherance to the wound surface, it should be tissue comparable, it should have an inner surface in contact with the wound surface that promotes the ingrowth of fibrovascular tissue, and/or it should provide protection from infection and prevention of fluid loss.
It is therefore an object of the present invention to provide cultured living skin equivalents that exhibit at least some of these features and which will substantially overcome, or ameliorate one or more of the abovementioned problems.